Inositol 1,4,5-trisphosphate (InsP3) is used as an intracellular messenger within a signalling pathway that serves to control many diverse cellular functions, including neurotransmitter and hormone responses, secretion, muscle contraction and phototransduction. Disorders of this signaling have been implicated in disorders including manic depressive illness, tumorigenesis and teratogenesis, and the relevance of this system to clinical studies will certainly grow as we come to understand it more. It is now well established that InsP3 functions principally by causing the liberation of Ca2+ ions sequestered within intracellular stores. However, recent improvements in techniques for monitoring intracellular Ca2+ have revealed great complexities in the patterns of InsP3-mediated Ca2+ liberation. Ca2+ is released as repetitive spikes or oscillations, and waves of Ca2+ release propagate through the cell. Furthermore, individual cells appear to contain many functionally independent Ca2+ stores, that each release their contents in a 'quantal', all-or-none manner. The spatial and temporal aspects of InsP3 signalling are undoubtedly important for the encoding of information by the InsP3-mediated transduction pathway. To study them we will employ Xenopus oocytes as a model system, as these cells have a well characterized InsP3 pathway, and their large size to obtain good spatial and temporal control of intracellular InsP3, and resolution of the resulting elevations in intracellular free Ca2 InsP3 will be formed in the cell by photolysis of a 'caged' precursor, and Ca2+ will be imaged by confocal video microscopy using long-wavelength indicator dyes. 'quantal' subcellular Ca2+ release units, to determine how their properties lead to the generation of repetitive Ca2+ spikes and propagating Ca2+ waves, and to see how information in the spatial and temporal patterns of Ca2+ liberation is encoded as changes in Ca2+-dependent membrane currents. High resolution imaging of Ca2+ evoked by photoreleased InsP3 will allow mapping of the distribution and morphology of subcellular InsP3-sensitive Ca2+ release sites, and their presence will be correlated with that of InsP3 receptors and endoplasmic reticulum marker proteins. At the whole cell level, we will also study the polarization of Ca2+ signalling between the two hemispheres of the oocyte. Functional studies of InsP3-evoked Ca2+ release at individual sites will elucidate the nature of the positive feedback that gives rise to regenerative Ca2+ release, and the roles of positive and negative feedback by Ca2+ will then be investigated in the generation of repetitive Ca2+ spikes, and propagating Ca2+ waves. Finally, the complex dependence of Ca2+-activated membrane C1- conductance on intracellular Ca2+ will be determined by simultaneous Ca2+ imaging and voltage-clamp recording.